| Hepatitis B virus (HBV) infection is a major global health burden currently affecting more than 350 million individuals worldwide. Interferon-alpha (IFN-?) is an approved medication for chronic hepatitis B. Gamma Interferon (IFN-?) is a key mediator of host innate and adaptive antiviral immunity against HBV infection in vivo. Interferon binds to the receptor and activates Jak-Stat signal pathway to induce the expression of Interferon stimulated genes (ISGs). Our aims were to elucidate the antiviral mechanism of interferons. The work can be divided into three parts.In the first part, IFN-?and IFN-?were verified to be able to inhibit HBV replication in human hepatocyte-derived cells. Also 38 IFN-stimulated genes (ISGs), which are highly inducible in hepatocytes, were tested for their ability to inhibit HBV replication upon overexpression in human hepatoma cells. As a result, overexpression of 8 ISGs reduced HBV DNA by more than 40%. Further study showed that 6 ISGs (including hZAP-S) mainly targeted on HBV RNA, while 2 other ISGs(APOBEC3G and IDO) targeted on HBV DNA, without affecting HBV RNA.In the second part, we focused on the mechanism study of IDO's anti-HBV effect. IDO, an IFN-?-induced enzyme catalyzing tryptophan degradation, efficiently reduced the level of intracellular HBV DNA, without altering the steady state level of viral RNA. Furthermore, expression of an enzymatically inactive IDO mutant did not inhibit HBV replication, and tryptophan supplementation in culture completely restored HBV replication in IDO-expressing cells, indicating tha the antiviral effect elicited by IDO is mediated by tryptophan deprivation. Interestingly, IDO mediated tryptophan deprivation preferentially inhibited viral protein translation and genome replication, but did not significantly alter global cellular protein synthesis. Finally, tryptophan supplementation was able to completely restore HBV replication in IFN-?, but not IFN-?, treated cells, which strongly argues that IDO is the primary mediator of IFN-? elicited antiviral response against HBV in human hepatocyte-derived cells.In the third part, we focused on the mechanism study of ZAP's anti-HBV effect. ZAP is an interferon induced host factor that limits the replication of a variety of RNA viruses. ZAP binds to the ZAP-responsive elements (ZRE) within viral RNA genome through zinc-finger motifs and recruits exosome to degrade the RNA substrates in cytoplasm. Our data demonstrated that human ZAP was endogenously expressed in hepatocyte derived cells and was induced by IFN-?or IPS-1, also hZAP dramatically reduced HBV pgRNA and subgenomic RNA in transfected hepatocyte derived cells. hZAP did not inhibit HBV core promoter activity in a luciferase reporter assay suggested that the viral RNA reduction is a posttranscriptional regulation. In contrast to the reported cytoplasmic retroviral RNA decay, hZAP mediated HBV RNA degradation occurs in the nucleus. Further study revealed that hZAP's antiviral effect is independent of the presence of viral core and polymerase. Similar to mouse ZAP, the C-terminal truncated hZAP retained antiviral activity, which indicated that the zinc-finger motif containing N-terminus is the antiviral functional domain of hZAP. Mutagenesis study revealed that the integrity of four putative zinc-finger motifs is essential for hZAP's antiviral against HBV, and the ZRE sequences were mapped at the terminal redundancy (nt 1820-1918) of HBV pgRNA. Finally siRNA study showed that endogenous hZAP is a host restriction factor of HBV replication; hZAP plays partial role in IPS-1 mediated HBV RNA decay. |
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